Our interest is in mechanisms that control chromosome replication and segregation. These basic mechanisms are widely shared among organisms and are fundamental to their growth and development. Subversion of the mechanisms causes aneuploidy, the hallmark of cancer cells. The importance of genome stability, therefore, can hardly be overstated. A greater understanding of the mechanisms operating in bacterial models should be of general interest and guide the rational development of therapeutics for diseases, particularly bacterial infections.We are studying the control mechanisms in E. coli and in V. cholerae, the latter having two chromosomes (chrI and chrII) provides an opportunity to study coordination of replication and segregation in a genetically tractable bacterium. In the last year, we have brought to closure a number of projects on replication control of a low copy number E. coli plasmid, P1. Nilangshu Das with help from a mathematician, Johan Paulsson (Cambridge U., UK), has characterized control-defective initiator mutants that confer higher copy number to the plasmid. The properties of the mutants are best explained assuming multiple modes of control involving transcriptional autorepression of the initiator gene, initiator inactivation by dimerization, and origin inactivation by pairing. The control is best when the three mechanisms cooperate. A new development in the field of E. coli DNA replication is the finding of additional binding sites for the initiator DnaA in the origin of replication, called I-sites (IHF protein-dependent DnaA binding sites). These sites recognize only ATP-bound DnaA. The generality of I-sites in DNA replication remains to be established as no consensus sequence could be derived from the three I-sites present in the E. coli origin. Richard Fekete has identified a possible I-site dependent replication origin in plasmid P1. The dependence of this origin on the ATP bound form of DnaA is being tested. Since ATP-DnaA level decreases after the initiation of chromosomal replication, this may be a way for the plasmid to correlate its replication with that of the chromosome and add a new paradigm in plasmid replication control.Knowing the importance for controlling initiator proteins in DNA replication control, Debasish Pal has studied the regulation of the RctB initiator for chrII replication. A strong promoter for the gene was identified and found to be autorepressed as well as regulated by unstream sequences and global regulators, IHF and Dam methylase. Increasing RctB in trans increased the copy number of a miniplasmid carrying the chrII origin, implying that RctB can be rate-limiting for chrII replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis. To study the regulation of replication of chrII, Tatiana Venkova-Canova has defined the origin and its negative control elements. The origin resembles those from iteron-type plasmids but the negative control locus is more extended and complex. It is already clear that the regulation of replication has diverged significantly from the ones operating in iteron-type plasmids or in E. coli.Unlike low copy number plasmids and many other bacteria, E. coli contains no homology to any known segregation system. To search for the E. coli centromere, Richard Fekete has labeled different loci on the same E. coli chromosome using LacI-YFP and Lambda cI-CFP fusion proteins bound to arrays of their respective binding sites. Analysis of migration of a few such pairs of loci using fluorescence microscopy suggested a potential centromere site since it migrated towards the cell pole ahead of the other loci, including the origin of replication.Preeti Srivastava is studying chromosome dynamics in V. cholera using fluorescence microscopy and flow cytometry.